Tumor necrosis factor-related apoptosis-inducing ligand (also named as TRAIL or Apo2L or TNFSF10 for short), which was discovered in 1995, is located on 3q26 of chromosome 3, and a member of tumor necrosis factor (TNF) superfamily. It is a type II transmembrane glycoprotein consisting of 281 amino acids. Its C-terminal is extracellularly located, and has sequence homologous to TNF family. TRAIL receptor 2 (also named as TRAIL-R2 or death receptor 5 or DR5) is widely expressed on the surface of a variety of cancer cells, but not or less expressed on the surface of normal cells. The extracellular region of TRAIL receptor 2 contains the binding region for its natural ligand TRAIL, and the intracellular region contains death domain. Once TRAIL or other agonists bind with DR5, DR5 homotrimerization occurs, and the death domains recruit intracellular relevant signaling proteins to form death-inducing signal complex (DISC), which triggers the downstream cell death signal transduction, thereby specifically induces the death of cancer cells. Previous studies have demonstrated that AD5-10, an agonistic mouse monoclonal antibody against human DR5, is capable of inducing the death of various cancer cells cultivated in vitro, and it also efficiently inhibits in vivo the formation and growth of human xenografted tumor in nude mice, while it is not toxic to normal tissues and cells (Guo Y et al., A novel anti-human DR5 monoclonal antibody with tumoricidal activity induces caspase-dependent and caspase-independent cell death. J Biol Chem 2005; 280 (51):41940-52; WO2006/017961, ZL200410070093.1). This suggests a good prospective clinical application for cancer therapy. However, as a heterogeneous immunoglobulin, murine monoclonal antibody would be recognized by human immune system after it is applied into human body, and then human anti-mouse antibody (HAMA) is generated and attenuates the therapeutic effect as well as possibly damage the human organs. Therefore, an important way to successfully apply murine antibody for therapy of human diseases is to humanize the murine monoclonal antibody, so as to reduce its immunogenicity to human body while maintaining the affinity, specificity and therapeutic efficacy.
The first generation of approach to engineer humanized antibody is to make human-murine chimeric antibody by linking the variable region of murine antibody to the constant region of human antibody. Even though the affinity of original murine antibody is well maintained, due to the spatial separation of the chimeric antibody variable and constant regions, the intact murine antibody variable region still may cause HAMA response. On the basis of chimeric antibodies, complementarity determining region (CDR) grafting technology (Williams D. G. et al., Humanising Antibodies by CDR Grafting in Antibody Engineering (2010) Vol. 1, p319-339, edited by Kontermann R. and Dübel S., Springer-Verlag Berlin Heidelberg) is further used to replace the relatively conserved framework regions (FR) of murine monoclonal antibody variable region with human FR region, and merely maintains the antigen-binding CDR region, so as to construct functional humanized monoclonal antibody. This makes it possible to minimize the immunogenicity of murine antibody molecule. Meanwhile, FR region not only functions as a framework supporting antibody molecules, but also involves in forming the correct conformation when antigen binds to antibody. Therefore, key amino acid residues in humanized antibody molecule FR region shall be subjected to back mutation to maximize the affinity and specificity of murine antibody. Further, since only several amino acid residues in heavy chain and light chain variable region complementarity determining regions (CDRs) and framework regions (FRs) are derived from the immunized animal species, all of the other parts are derived from human same isotype antibody, this minimizes human response against mouse antibody while maintaining specificity and affinity of murine monoclonal antibody, prolongs its half-life in vivo and improves its pharmacokinetic properties. Furthermore, humanized antibody has effector functions of human antibody, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDCC), thus is more conducive to clinical application.
Mammalian cell expression system possesses intact transcriptional, translational and post-translational processing as well as secretory mechanism, and can efficiently produce an intact antibody molecule with correct folding, assembly, spatial conformation and good biological activity. Such cell expression system is ideal system for humanized antibody production. Thus, the construction of eukaryotic expression vector is very crucial for increasing expression level of humanized antibody. An ideal eukaryotic expression vector needs a strong promoter and a proper selectable marker, which benefit not only the amplification of gene itself, but also the selection of positive cells.
Therefore, the technical purpose of present invention is to produce a novel humanized monoclonal antibody against human DR5 and the eukaryotic expression vector thereof, as well as the use of said humanized monoclonal antibody in the treatment of various cancers.